U.S. patent number 5,372,784 [Application Number 08/212,674] was granted by the patent office on 1994-12-13 for measurement of bacterial co.sub.2 production in an isolated fluorophore by monitoring an absorbance regulated change of fluorescence.
This patent grant is currently assigned to Baxter Diagnostics Inc.. Invention is credited to Shoshana Bascomb, Jamie Bobolis, Roger J. Morris, Carolyn S. Olson, David Sherman.
United States Patent |
5,372,784 |
Morris , et al. |
December 13, 1994 |
Measurement of bacterial CO.sub.2 production in an isolated
fluorophore by monitoring an absorbance regulated change of
fluorescence
Abstract
A multi-layer blood culture sensor includes two matrices. The
first matrix is a polymer that is permeable to carbon dioxide and
water, but impermeable to protons. A pH sensitive absorbance based
dye is encapsulated or isolated in the polymer. The second matrix
is a polymer with a pH insensitive fluorescent dye encapsulated or
isolated in the polymer. The matrices are spectrally coupled and
are useful for the determination of microorganisms in a blood
culture bottle.
Inventors: |
Morris; Roger J. (Sacramento,
CA), Bascomb; Shoshana (Davis, CA), Olson; Carolyn S.
(Sacramento, CA), Bobolis; Jamie (Sacramento, CA),
Sherman; David (Sacramento, CA) |
Assignee: |
Baxter Diagnostics Inc.
(Deerfield, IL)
|
Family
ID: |
27399140 |
Appl.
No.: |
08/212,674 |
Filed: |
March 11, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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638481 |
Jan 4, 1991 |
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609278 |
Nov 5, 1990 |
5173434 |
Dec 22, 1992 |
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238710 |
Aug 31, 1988 |
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Current U.S.
Class: |
435/287.9;
422/504; 422/52; 422/82.05; 422/82.07; 422/82.08; 435/288.7;
435/34; 435/39; 435/808; 436/167; 436/169; 436/170; 436/172 |
Current CPC
Class: |
C12Q
1/06 (20130101); G01N 21/17 (20130101); G01N
21/272 (20130101); G01N 21/78 (20130101); G01N
21/80 (20130101); G01N 21/534 (20130101); G01N
2201/122 (20130101); Y10S 435/808 (20130101) |
Current International
Class: |
C12M
1/34 (20060101); C12Q 1/06 (20060101); G01N
21/25 (20060101); G01N 21/17 (20060101); G01N
21/77 (20060101); G01N 21/27 (20060101); G01N
21/78 (20060101); G01N 21/80 (20060101); G01N
21/47 (20060101); G01N 21/53 (20060101); G01N
021/76 () |
Field of
Search: |
;422/55,56,52,82.05,82.07,82.08 ;435/34,39,808,291
;436/167,169,170,172,800 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0091837 |
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Apr 1984 |
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EP |
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0104463 |
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Apr 1984 |
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EP |
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0124193 |
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Nov 1984 |
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EP |
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0171158 |
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Feb 1986 |
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EP |
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1601689 |
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Nov 1981 |
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GB |
|
Other References
Ando et al., Pyruvate as a Fluorescence Quencher: A New
Spectroscopic Assay for Pyruvate Reactions, Anal. Bio. 129, 170-175
(1983). .
Shoshana Bascomb, Enzyme Tests in Bacterial Identification, Method
in Microbiology, 19: 105 (1987). .
Blumberg et al., Hemoglobin Determined in 15 .mu.L of Whole Blood
by "Front-Face" Fluorometry, Clinical Chemistry vol. 26/3 pp.
409-413 (1980). .
Carmel et al., An Intramolecularly Quenched Fluorescent Tripeptide
as a Flurogenic Substrate of Angiotensin-I-Converting Enzyme and of
Bacterial Dipeptidyl Carboxypeptidase, Eur. J. Biochem., 87 265-273
(1978). .
Carmel et al., Intramolecularly-Quenched Fluorescent Tripeptide as
a Fluorogenic Substrates of Leucine Aminopeptidase and Inhibitors
of Clostridial Aminopeptidase, Eur. J. Biochem., 73, 617-625
(1977). .
Fleminger et al., Fluorogenic Substrates for Bacterial
Aminopeptidase P and Its Analogs Detected in Human Serum and Calf
Lung, Eur. J. Biochem., 125, 609-615 (1982). .
Florentin et al., A Highly Sensitive Fluorometric Assay for
"Enkephalinase," a Neutral Metalloendopeptidase That Releases
Tyrosine-Glycine-Glycine from Enkephalins, Anal. Biochem. 141,
62-69 (1984). .
Rhines et al., Simplex Optimization of a Fiber-Optic Ammonia Sensor
Based on Multiple Indicators, Anal. Chem., 60, 76-81 (1988). .
Wolfbeis et al., Fibre-Optic Fluorescing Sensor for Ammonia, Anal.
Chimica Acta, 185, 321-327 (1986). .
Yaron et al., Intramolecularly Quenched Fluorogenic Substrates for
Hydrolytic Enzymes, Anal. Biochem., 95, 228-235 (1979)..
|
Primary Examiner: Housel; James C.
Assistant Examiner: Wallenhorst; Maureen M.
Attorney, Agent or Firm: Buonaiuto; Mark J. Tymeson; Cynthia
G. Flattery; Paul C.
Parent Case Text
This application is a continuation of application Ser. No.
07/638,481, filed on Jan. 4, 1991, now abandoned, which is a
continuation-in-part of application Ser. No. 07/609,278, filed on
Nov. 5, 1990 and now U.S. Pat. No. 5,173,434 issued on Dec. 22,
1992, as well as a continuation-in-part of application Ser. No.
07/238,710, filed on Aug. 31, 1988 and now abandoned.
Claims
What is claimed is:
1. A multi-layer sensor for determining the concentration or
presence of a microorganism in a bodily fluid which comprises:
a. a pH sensitive absorbance based dye encapsulated in a first
light transmissive, gas permeable, proton impermeable matrix;
and
b. a pH insensitive fluorescence dye encapsulated in an inert light
transparent second matrix, wherein said first and second matrices
are spectrally coupled and in close proximity.
2. The multi-layer sensor of claim 1 wherein said pH sensitive
absorbance based dye is selected from the group consisting of
xylenol blue and bromothymol blue.
3. The multi-layer sensor of claim 1 wherein said fluorescence dye
is selected from the group consisting of rhodamine 101 and
rhodamine B.
4. The multi-layer sensor of claim 1 wherein said first and second
matrices are selected from the group consisting of silicone and
acrylic.
Description
FIELD OF THE INVENTION
This invention relates to a method to detect the presence or
determine the concentration of microorganisms in a solution by
regulating light reaching or emitted by a fluorophore encapsulated
in a chemically inert light transparent matrix.
BACKGROUND OF THE INVENTION
Microorganisms present in bodily fluid can be detected using a
culture bottle. Generally, a culture bottle is a flask allowing
positive cultures to be detected rapidly. The flask is generally a
transparent closed container filled with nutrient that promotes the
growth of the organism. In particular, bacteria in blood can be
detected in culture. U.S. Pat. No. 4,772,558 (Hammann).
Many different qualitative and quantitative detection means are
used to monitor the growth of microorganisms in a culture bottle.
The microorganisms in a culture bottle have been detected by use of
external detectors such as a magnifying lens, U.S. Pat. No.
4,543,907 (Freudlich). Additionally, integral detectors such as
liquid level indicators can show bacterial growth as a function of
increased pressure in the vessel V. Swaine et al., EPA 124,193.
Additionally, microorganisms can be detected by measuring changes
in pH caused by bacterial growth, Mariel, G.B. Patent No.
1,601,689.
Still another method to detect microorganisms involves the use of a
culture media that contains a compound which changes color or
appearance according to the growth of microorganisms. The change in
the media can be detected with a spectrophotometer. There are many
examples of reactions used in Microbiology that rely on a color
change. Bascomb, Enzyme Tests in Bacterial Identification, 19 Meth.
Microbio. 105 (1987). For example. a variety of organisms can be
classified in large part by their pattern of fermentation,
oxidation or assimilation of carbon sources. Fermentation of
carbohydrates results in the production of acid which causes a
decrease in pH. This drop in pH can be easily detected by including
a pH indicator like bromothymol blue or phenol red. With both
indicators, acid conditions representing the fermentation of a
particular carbohydrate result in a yellow color (changing from
blue-green for bromothymol blue or pink/red for phenol red). The
same approach can be adopted for a variety of carbohydrates,
ranging from monosaccharides like glucose to polysaccharides like
inulin. In an analogous fashion, increasing pH can also be
monitored. Assays for detecting the presence of decarboxylase and
urease, and the ability to use malonate are based on an increase in
pH, as indicated by a color change in the indicator. Turner, et al.
U.S. Pat. No. 4,945,060 discloses a device for detecting
microorganisms. In this device changes in the indicator medium
resulting from pH changes in CO.sub.2 concentration in the medium
are detected from outside the vessel.
Chemical and enzymatic reactions are used to detect or quantirate
the presence of certain substances in microbiological or other
assays. Many of these tests rely on the development or change of
color or fluorescence to indicate the presence or quantity of the
substance of interest.
Another approach to determine if an organism can degrade a
particular substrate, is to use a reagent which is capable of
reacting with one or more of the intermediates or final products.
For example, the detection of the reduction of nitrate to nitrite.
If nitrite is formed, then a pink to deep red color will result
when sulfanilic acid and alpha-naphthylamine are added to the
reaction mixture.
In contrast to the indirect detection of an enzymatic reaction
illustrated by the nitrate/nitrite test, it is possible to use a
synthetic analog of a natural substrate to directly indicate the
presence of an enzyme. For example, methylene blue can be reduced
under certain conditions by the action of reductase, resulting in a
shift from blue to colorless. In another test, the oxidase assay
relies on the interaction of cytochrome oxidase with N, N, N',
N'-tetramethyl-p-phenylenediamine producing a blue color.
Another example is the ability of microorganisms to degrade
sulfur-containing amino acids as indicated by the production of
H.sub.2 S. Typically, the organism is incubated with a high
concentration of a sulfur-containing substrate (e.g. cysteine,
cystinc) in an acid environment. The production of H.sub.2 S is
indicated by the formation of a black precipitate in the presence
of ferric ammonium citrate.
Enzymes can usually act on more than one substrate. This allows for
the use of synthetic enzyme substrates for the detection of enzyme
activities. Synthetic substrates contain a metabolic moiety
conjugated with a chromatic or fluorescent moiety. The conjugated
molecule usually has a different absorption and/or emission
spectrum from the unconjugated form. Moreover, the unconjugated
chromatic or fluorescent moiety shows a considerably higher
absorption or fluorescence coefficients than those of the
conjugated molecule. This allows the measurement of small amounts
of products of enzyme activities in the presence of the large
amounts of conjugated substrate required for maximal enzyme
activity. An example of a synthetic enzyme substrate is
o-nitro-phenol-.beta.-galactopyranoside used for the detection of
activity of the enzyme .beta.-galactosidase. The conjugated
substrate is colorless. The .beta.-galactosidase enzyme hydrolyzes
the substrate to yield .beta.-galactosidase and o-nitrophenol.
o-nitro-phenol absorbs strongly at 405 nm, and its release can be
measured by the increase in absorbance at that wavelength. Bascomb,
Enzyme Tests in Bacterial Identification, Meth. Microbiol. 19, 105
(1987), reviewed the synthetic moieties used for enzyme substrates
and the enzymatic activities measurable using this principle.
Presently, the monitoring of color or color end-product in chemical
and microbial reactions is usually achieved in either of two ways;
1) the detection of color or color end-product can be achieved by
visual observation and estimated qualitatively, or 2) the detection
of color end-products or loss of color can be achieved by measuring
the intensity of color instrumentally. Spectrophotometers that
measure light absorbance are commonly used for this purpose. When
measuring the concentration of a number of substances it is
advantageous to use one instrument based on one principle of
measurement, otherwise cost increased.
Although the use of colorimetric reactions is widespread there are
limitations, especially in the sensitivity of detection. In order
to improve sensitivity and, in the case of identification of
microorganisms, thereby to decrease the time required to obtain a
result, fluorescence-based methods frequently are used.
Unfortunately, it may not be possible to develop a fluorescent
equivalent to every assay. Additionally, the fluorescent reagents
themselves may be highly toxic and therefore difficult to
commercialize.
In such cases one might need to measure activities of some enzymes
fluorometrically, the others colorimetrically. However, most
instruments are suited to measure either absorbance or
fluorescence, and very few can be used to measure both.
The general principle of fluorescence quenching has been accepted
as a way to detect or determine enzymatic or chemical reactions.
For example, Fleminger et al. synthesized intramolecularly quenched
fluorogenic substrates for the assay of bacterial aminopeptidase,
P. Fleminger et al., Fluorogenic Substrates for Bacterial
Aminopeptidase P and its Analogs Detected in Human Serum and Calf
Lung, Eur. J. Blochem. 125, 609 (1982). In this case, the
fluorescence of the aminobenzoyl group is quenched by the presence
of a nitrophenylalanyl group. When the enzyme is present, the
nitrophenylalanyl group is cleaved, with a concommitant increase in
the sample's fluorescence. A variety of enzymes have been assayed
by this type of procedure, including hydrolytic enzymes, other
amino- and carboxypeptidases and an endopeptidase. Yaron et al.,
Intramolecularly Quenched Fluorogenic Substrates for Hydrolytic
Enzymes, Anal. Bioche. 95, 228 (1979); Carmel et al.,
Intramolecularly--Quenched Fluorescent Peptides as Flurorogenic
Substrates of Leucine Aminopeptidase and Inhibitors of Clostridial
Aminopeptidase, Eur. J. Biochem. 73, 617 (1977); Carmel et al., An
Intramolecularly Quenched Fluorescent Tripeptide as a Fluorgenic
Substrate of Angiotensin-I-Converting Enzyme and of Bacterial
Dipeptidyl Carboxypeptidase, Eur. J. Biochem. 87, 265 (1978);
Florentin et al., A Highly Sensitive Fluorometric Assay for
"Enkephalinase", a Neutral Metalloendopeptidase that Releases
Tyrosine-Glycine-Glycine from Enkephalins, Anal. Biochem 141, 62
(1984). In each of the previous approaches, a synthetic substrate
containing a quenching group and a fluorescing group was generated
in order to detect the activity of the enzyme.
An alternative to this approach would involve the synthesis of a
resonance energy transfer pair of fluorescing groups on a substrate
molecule. In this method, cleavage by the enzyme of one of the
groups would result in a decrease in fluorescence, since the
critical distance would be exceeded, eliminating the transfer of
energy. However, the previously discussed approaches are limited to
specifically designed substrates.
Still another approach involves the estimation of a chromophore by
fluorescence measurement. See W. Blumberg et el., Hemoglobin
Determined in Whole Blood "Front Face" Fluorometry, Clin. Hemo. 26,
409 (1980). Blumberg disclosed an assay based on attenuation of
fluorescence of a dye, whose excitation wavelengths overlap with
the absorption wavelengths of the chromophore.
Subsequently, M. Shaffar, U.S. Pat. No. 4.495,293 (hereinafter
Shaffar) filed a patent application disclosing a method to
fluorometrically determine a ligand in an assay solution using
conventional fluorometric techniques. In Shaffer the intensity of
the fluorescence emitted by the assay solution is related to the
change in transmissive properties of the assay solution produced by
the interaction of the ligand to be determined and a reagent system
capable of producing change in the transmissive properties of the
assay solution in the presence of the ligand. More particularly,
Shaffar discloses a method to monitor absorbance using a
fluorophore in solution with the chromophore. In this method the
fluorophore may interact with the assay cocktail and produce
changes in fluorescence intensity which are unrelated to the change
being measured. The selection of the fluorophores is also
restricted, in that pH dependent or environment sensitive
fluorophores cannot be utilized. Additionally, when the fluorophore
is in solution, less than accurate measure of absorbance may be
obtained because light is absorbed exponentially through the
chromophore sample.
Similarly, Beggs & Sand, EPA 91,837 disclosed a solution based
method for determination of tryptophan-deaminase activity by
measuring the reduction in fluorescence in the presence of a
chromophore produced by the interaction between indole pyruvic acid
and metal ions using a fluorophore "whose fluorescence is capable
of being quenched by the indole pyruvate-metal ion complex, the
ions of the fluorophore being present throughout the incubation
period".
Also, Sands, U.S. Pat. No. 4,798,788 discloses a process to detect
a nitrate reducing microorganism by measuring reduction of
fluorescence in solution by causing the diazotization of the
fluorophore. In all these cases a specific fluorophore needs to be
chosen for each test to ensure that it will fluoresce under the
conditions of the test, e.g. only few fluorophores fluoresce at pH
of less than 2.0.
SUMMARY OF THE INVENTION
This invention provides a method to use a fluorophore encapsulated
in a chemically inert matrix which is transparent at the
wavelengths of interest. The fluorophore, positioned to intersect
the transmission light path, indirectly monitors absorbance or
changes in the absorbance of a chromophore encapsulated or isolated
by a gas permeable polymetric matrix. The use of a fluorophore
encapsulated in or isolated by a matrix allows for the sequential
influence of reaction components on the intensity of light
detected. This result can be achieved when the absorption spectrum
of a chromophore overlaps the excitation and/or the emission
spectrum of a fluorophore, thereby allowing the change in
fluorescence to be related to the intensity of color in the
reaction and consequently related to the quantity of the substance
of interest. It should be noted that the spectrum is not limited to
visible light.
More particularly, this invention relates to a multi-layer body
fluid culture sensor comprised of a pH sensitive absorbance based
dye spectrally coupled to a pH insensitive, or pH sensitive dye
that is highly buffered, fluorescence based dye. The pH sensitive
absorbance based dye is encapsulated or isolated by a polymeric
layer that is permeable to CO.sub.2 and water, but impermeable to
protons. The pH insensitive fluorophore is encapsulated or isolated
in the second polymeric layer that may or may not be permeable to
CO.sub.2 and water. This type of sensor may be used to detect or
determine the concentration of microorganisms in bodily fluid. The
spectral criterion required to make this determination are such
that the absorption spectrum of the chromophore must overlap the
excitation and/or emission spectrum of the fluorophore, thereby
allowing the change in fluorescence to be related to the change in
the reaction and consequently related to the presence or quantity
of the substance of interest.
Further, this sensor is used to monitor microbial infections grown
in a fluid culture bottle. In particular, this sensor can be used
to monitor bacterial growth. As bacteria grow they generate
CO.sub.2. The CO.sub.2 generated by the bacteria diffuses into the
polymeric layer that is in direct contact with a hydrated pH
sensitive absorbance based dye. The CO.sub.2 reacts with the
aqueous environment to form carbonic acid (H.sub.2 CO.sub.3), which
lowers the pH of the absorbance dye environment. This results in a
concomitant change in the pH sensitive spectrum of the dye.
Typically, as the absorbance of an absorbance based dye decreases
more light reaches the fluorophore for excitation which results in
a larger amount of emitted fluorescence.
In one embodiment the gas permeable, proton impermeable polymeric
matrix is silicone. Additionally, in one embodiment of this
invention a detector, such as a photomultiplier tube, is placed
under the blood culture bottle to detect fluorescent emission.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a schematic diagram of a multi-layer blood culture
sensor.
FIG. 2 shows a blood culture growth curve detected by a xylenol
blue-rhodamine 101 sensor.
FIG. 3 shows a blood culture growth curve detected by xylenol blue
in silicone-rhodamine B in acrylic sensor.
FIG. 4 shows a blood culture growth curve for a xylenol blue in
silicone-6213 acrylic sensor.
FIG. 5 shows a blood culture growth curve for a bromothymol blue in
silicone-rhodamine 101 in silicone sensor.
DETAILED DESCRIPTION - BEST MODE
In this approach, fluorescence from a fluorophore embedded in a
chemically inert light-transparent matrix, is modulated by a pH
sensitive absorbance dye encapsulated in a polymeric gas permeable,
but proton impermeable matrix. The assay is carried out in a blood
culture bottle.
In a fluorometric based colorimetric assay the fluorescence
intensity is regulated by changes in absorbance of an interfering
chromophore. As a pH change occurs the chromophoric material alters
the amount of light reaching the fluorophore and/or the amount of
emitted light reaching the detector. Spectrally compatible
colorimetric and fluorometric indicators are selected so that as
the pH changes due to the production of CO.sub.2 by microorganisms
present in the sample, the colorimetric indicators regulate the
amount of light reaching the fluorophore and/or photodetector and,
thus cause a change in the amount of emitted light from the
fluorescent dye received by the photodetector. This change is
detected with a fluorescent reader and can be correlated with the
presence-or concentration of microorganism in the sample.
A bodily fluid culture sensor is comprised of a pH sensitive
absorbance based dye encapsulated in or isolated by a polymeric gas
permeable, but proton impermeable matrix and a fluorescent dye in a
second polymeric matrix.
Spectrally compatible colorimetric and fluorometric indicators are
selected so that when an organism is present in a sample, the
colorimetric indicator will regulate the amount of light reaching
the fluorophore thereby causing a change in the emission intensity
from the fluorescence dye reaching the photodetector. The change,
indicating the presence of bacteria, is detected with a
fluorometric reader.
More particularly, spectrally compatible fluorescence and
absorbance dyes are selected so that as carbonic acid (H.sub.2
CO.sub.3) is produced, the absorbance of the dye will change
thereby regulating the amount of light reaching the fluorophore
and/or photodetector, thus producing a change in the measured
fluorescence. This change is detected with a fluorescence reader.
Spectrally compatible dyes are xylenol blue and rhodamine b.
Additionally, bromothymol blue and rhodamine 101 are also
spectrally compatible.
Thus, in practice a culture bottle containing the appropriate
growth media can be inoculated with E. coli. As the organism grows,
it produces CO.sub.2 gas. The silicone is permeable to the
CO.sub.2. The GO.sub.2 diffuses into the absorbance layer and
reacts with water to produce carbonic acid (H.sub.2 CO.sub.3). The
carbonic acid causes a drop in the pH in the absorbance dye
environment resulting in a change in measured absorbance. For
example, as the pH drops in an absorbance layer containing the dye
xylenol blue, the absorbance of xylenol blue decreases, thereby
allowing more light to reach the fluorophore to excite it and thus
increase the amount of fluorescence emitted at 590 nm. A positive
culture using xylenol blue as the absorbance dye is detected by a
measured increase in fluorescence as the xylenol blue decreases in
absorbance.
The pH sensitive absorbance based dye is encapsulated in or
isolated by a polymeric matrix that is gas permeable, but proton
impermeable. The polymeric matrix must be optically transparent in
the visible region, permeable to gas, autoclavable, stable for at
least six months, and proton impermeable. In particular, silicone
may function as the polymeric matrix used to encapsulate or isolate
the absorbance based dye. Silicones found to meet these criteria
were Dow, Rhone Poulenc, G. E. and Wacker.
Similarly, the fluorescence based dyes can also be encapsulated in
a polymeric matrix. The polymeric matrix used for the fluorophore
does not have to meet all of the above requirements listed for the
matrix used to encapsulate or isolate the absorbance dye. The
similar features that it must possess are that it must be optically
transparent in the visible region, autoclavable and stable for at
least six months.
The polymeric matrix containing or isolating the absorbance based
dye must be coupled to the polymeric matrix containing the
fluorescent dye. It should be noted that the polymeric matrices
must be in close proximity so that light that has been regulated by
the absorbance layer will have an effect on the emission intensity
of the fluorophore as received by the photodetector. This can be
accomplished by applying the same polymeric material to one side of
each polymeric matrix and curing these matrices. Once the matrices
containing the dyes have been adhered together they must be
rehydrated. The clarity of the sensor upon rehydration is also a
factor in matrix selection.
In the present invention, a bodily fluid culture sensor, FIG. 1, is
comprised of a pH sensitive absorbance based dye encapsulated in or
isolated by a polymeric gas permeable, but proton impermeable
matrix 4 and a fluorescent dye in a second polymeric matrix 2.
Reflective surface 6 can be included to facilitate the transmission
of light to the detecting element 12. In FIG. 1 interrogation light
enters the sensor and is regulated by pH sensitive matrix 4 which
in turn causes a change in the fluorescence emission 10 of the
fluorophore in matrix 2. This sensor offers the advantage of
maximal surface area.
In an alternative embodiment, an acrylic encapsulated fluorophore
or silicone embedded fluorescence material is adhered to an
absorbance dye isolating polymeric layer, to make a two layer
sensor.
In another embodiment, both the fluorescence and absorbance
embedded material are poured into blood culture bottles. In this
embodiment the fluorophore embedded silicone material is poured on
top of absorbance embedded silicone.
The optical interrogation system comprises a visible output,
400-700 nm, light source focused onto one end of a bifurcated fiber
optic cable. The common end is positioned close to the sensor,
while the other end is positioned close to a photodetector,
typically a photomultiplier tube. Appropriate excitation and
emission filters are used to select wavelengths of choice for each
dye. A beam splitter is used to divert a portion of the excitation
light to a second photodetector and acts as a reference. A
photodetector converts light to a current source which is converted
to a voltage using an operational amplifier. A 12 bit analog to
digital conversion offers sufficient dynamic range to read the
voltage. A computer program is then used to read, plot and store
data.
A measurement is taken by first reading reference light intensity.
Next the reading from the sensor disk is measured. The data is
plotted by taking the ratio of reference, excitation light, to
sample. In particular, as CO.sub.2 levels increase in the blood
culture bottle, the absorbance of the absorbance dye changes,
thereby changing the amount of light reaching the fluorescence
layer and/or photodetector. This causes a change in emitted
fluorescence that is detected.
The following examples serve to illustrate the method of the
present invention. The concentration of reagents and other variable
parameters are only shown to exemplify the methods of the present
invention and are not to be considered limitations thereof.
EXAMPLE 1 XYLENOL BLUE--RHODAMINE 101 SENSOR
Wacker silicone elastomer 3601 part A is thoroughly mixed with
Wacker 3601 catalyst part B in a 9:1 ratio, as recommended by the
manufacturer. Next 5% w/w of a 50 mM xylenol blue, dissolved in 5
mM borate buffer pH 11 containing 1% Tween 80, is added to the
silicone and homogenized to ensure a uniform distribution of the
dye. The absorbance layer mixture is then poured into an aluminum
square mold to a thickness of 30/1000 of an inch and cured at
55.degree. C. for 2 hours.
Wacker silicone is prepared, as described above. Next 2% w/w of 7.5
mM Rhodamine 101, in 50 mM Tris-HCl buffer DH 8.5 in 95% ethylene
glycol, is added to the silicone. The mixture is poured over the
previously cured xylenol blue layer in the mold, described above,
and cured at 55.degree. C. overnight. This cured. dehydrated,
double layer sensor consists of two distinct layers, each 30/1000
of an inch thick. Disks may now be punched out of the mold and
adhered onto the base of bottles using more silicone, ensuring that
the absorbance layer is face down. Finally, the bottles are cured
at 55.degree. C. for 15 minutes, rehydrated with normal saline and
autoclaved on the wet cycle for 17 minutes. Saline is replaced with
growth media and inoculated with E. coli by injecting a suspension
with a sterile needle through the septum. The blood culture bottle
is placed in the instrument and fluorescence emission is
measured.
As the concentration of CO.sub.2 increases in the blood culture
bottle, the absorbance of the pH sensitive absorbance dye, xylenol
blue, decreases, thus allowing more light to reach the fluorophore,
rhodamine 101, to thus increase the amount of fluorescence emitted
at 590 nM. This increase in fluorescence intensity v. time is shown
in the blood culture growth curve at FIG. 2.
EXAMPLE 2 XYLENOL BLUE IN SILICONE/RHODAMINE B IN ACRYLIC
Rhone Poulenc silicone elastomer 141 part A is thoroughly mixed
with Rhone Poulenc 141 catalyst part B in a 10:1 ratio, as
recommended by the manufacturer. Next 1% w/w of a 100 mM xylenol
blue solution pH 11, dissolved in 10 mM borate buffer containing 1%
Tween 80, is added to the silicone and mixed thoroughly with a
tongue blade to ensure uniform distribution of the dye. The
absorbance layer mixture is then poured into an aluminum square
mold to a thickness of 30/1000 of an inch. The mold is allowed to
sit out on the countertop at room temperature for about one hour or
until the bubbles have disappeared, at which time the mold is
placed in the incubator to cure at 55.degree. C. for two hours.
Rhone-Poulenc silicone is prepared, as described above. Next, a
40/1,000" thick acrylic disc (Glasflex, Inc.). approximately 1 cm
in diameter, containing 0.2 grams/lb of rhodamine B (Sigma) is
glued onto the above absorbance layer using the Rhone-Poulenc
silicone at the 10/1 ratio as glue. The double layer sensor is then
placed back in the 55.degree. C. incubator for two hours to allow
for adherence of the two layers. Following the curing, the double
layer sensor is punched out with a cork borer, and glued onto the
base of a Wheaton bottle, ensuring that the absorbance layer is
face down, using the Rhone Poulenc silicone as mentioned above. The
bottle is placed in the 55.degree. C. incubator to cure for at
least two hours. The bottle is then rehydrated overnite and tested
the following day as described in Example 1.
As the concentration of CO.sub.2 increases in the blood culture
bottle, the absorbance of the pH sensitive absorbance based dye
xylenol blue decreases, thus allowing more light to reach the
fluorophore (rhodamine B) doped acrylic, to thus increase the
amount of fluorescence emitted at 590 nm. This increase in
fluorescence intensity v. time is shown in the blood culture growth
curve in FIG. 3.
EXAMPLE 3 XYLENOL BLUE IN SILICONE/6213 RED STANDARD ACRYLIC
Wacker silicone elastomer 3601 part A is thoroughly mixed with
Wacker 3601 catalyst part B in a 9:1 ratio, as recommended by the
manufacturer. Next 5% w/w of a 50 mM xylenol blue, dissolved in 5
mM borate buffer pH 11 containing 1% Tween 80, is added to the
silicone and homogenized to ensure a uniform distribution of the
dye. The absorbance layer mixture is then poured into an aluminum
square mold to a thickness of 30/1000 of an inch and cured at
55.degree. C. for two hours.
Next, a 40/1,000" thick acrylic disc (Gilasflex, approximately 1 cm
in diameter, referred to as No. 62l3 Red (Glassflex Standard
Product) is glued onto the above absorbance layer using the Wacker
silicone at the 9/1 ratio as glue. The double layer sensor is then
placed back in the 55.degree. C. incubator for two hours to allow
for adherence of the two layers. Following the curing, the double
layer sensor is punched out with a cork borer, and glued onto the
base of a Wheaton bottle, ensuring that the absorbance layer is
face down, using the Rhone Poulenc silicone as mentioned above. The
bottle is placed in the 55.degree. C. incubator to cure for at
least two hours. The bottle is then rehydrated overnite and tested
the following day as described in Example 1.
As the concentration of CO.sub.2 increases in the blood culture
bottle, the absorbance of the pH sensitive absorbance based dye
xylenol blue decreases, thus allowing more light to reach the
fluorophore (rhodamine b) doped acrylic, to thus increase the
amount of fluorescence emitted at 590 nm. This increase in
fluorescence intensity v. time is shown in the blood culture growth
curve in FIG. 4.
EXAMPLE 4 BROMOTHYMOL BLUE IN SILICONE/RHODAMINE 101 IN
SILICONE
Wacker silicone elastomer 3601 part A is thoroughly mixed with
Wacker 3601 catalyst part B in a 9:1 ratio, as recommended by the
manufacturer. Next 5% w/w of 50 mM bromythymol blue, dissolved in 5
mM tris buffer pH 12 in ethylene glycol, is added to the silicone
and homogenized to ensure a uniform distribution of the dye. The
absorbance layer mixture is then poured into an aluminum square
mold to a thickness of 30/1000 of an inch and cured at 55.degree.
C. for two hours.
Wacker silicone is prepared, as described above. Next 2% w/w of 7.5
mM Rhodamine 101, in 50 mM Tris-HCl buffer pH 8.5 in 95% ethylene
glycol, is added to the silicone. The mixture is poured over the
previously cured xylenol blue layer in the mold, described above to
isolate the absorbance layer. This sensor is then cured at
55.degree. C. overnight. This cured, dehydrated, double layer
sensor consists of two distinct layers, each 30/1000 of an inch
thick. Disks may now be punched out of the mold and adhered onto
the base of bottles using more silicone, ensuring that the
absorbance layer is face down. Finally, the bottles are cured at
55.degree. C. for 15 minutes, rehydrated with normal saline and
autoclaved on the wet cycle for 17 minutes. Saline is replaced with
growth media and inoculated with E. coli by injecting a suspension
with a sterile needle through the septum. The blood culture bottle
is placed in the instrument and fluorescence emission is measured.
The increase in fluorescence intensity v. time is shown in blood
culture growth curve in FIG. 5.
Although this invention has been described with respect to specific
embodiments, the details thereof are not to be construed as
limitations, for it will be apparent that various equivalents,
changes and modifications may be resorted to without departing from
the spirit and scope thereof and it is understood that such
equivalent embodiments are intended to be included herein.
* * * * *